In recent years advances in recombinant DNA biotechnology have made it possible to produce a wide variety of useful polypeptide products in host cells which have been transformed and transfected with DNA sequences which code for production of the polypeptide products. Thus hormones such as insulin and growth hormones e.g. human growth hormone, and industrially or therapeutically useful enzymes, such as chymosin and tissue plasminogen activator (tPA) have been produced in transformed host cells.
Bacterial cells, in particular E. coli, have been used as host cells for the production of recombinant polypeptide products. The genetic systems of such bacterial cells are now relatively well understood and also such cells exhibit good growth characteristics. However, when such bacterial cells are used to overproduce foreign proteins, the foreign products typically accumulate within the host cells and it is usually necessary to disrupt the cells to effect recovery of the products. Also recombinant products are often produced within bacterial host cells in the form of insoluble aggregates in which the polypeptides are not in their native, biologically functional form. It is necessary, therefore, to solubilise and denature/renature the insoluble polypeptide products to obtain useful products in soluble, native, biologically functional form. For instance, British Patent No. GB 2100732B describes inter alia processes for production of methionine-prochymosin in E. coli involving disruption of the bacterial host cells and treatment with urea or guanidine HCl to solubilise the unnatural prochymosin-containing aggregates which are produced. The processes of cell disruption and denaturation/renaturation add significantly to the cost of producing recombinant polypeptide products.
Attempts have been made therefore to develop bacterial expression systems which secrete recombinant products into the extracellular culture medium. For example, recombinant heterologous polypeptides have been expressed in bacteria as fusion proteins in which the heterologous polypeptide sequence is joined with an N-terminal signal sequence. However, such fusion proteins, although exported across the inner membrane in Gram-negative bacteria with concomitant removal of the signal sequence, fail to cross the outer membrane and remain within the periplasm. Thus, it is still necessary to disrupt the host cells to effect recovery of heterologous recombinant products and denaturation/renaturation treatment may be required to yield products in native, biologically functional form.
Also `leaky` mutants of Gram-negative bacterial host cells such as E. coli have been proposed for use in the production and secretion of products to the extracellular medium. However, such mutant cells are often not suitable for large scale production of heterologous protein products since the yield of product is generally low and the fragility of the cells makes them unsuitable for growing on a large scale.
Haemolysin (Hly) is an extracellular protein toxin which is produced by some strains of E. coli, and as such is one of the few proteins produced by Gram-negative bacteria which are transported across both the cytoplasmic and outer membranes. Hitherto, however, the processes by which haemolysin is secreted to the extracellular medium have not been satisfactorily explained. Studies have indicated that a specific transport system determined by two of the hly genes is responsible for transport of haemolysin across the outer membrane (Wagner et al, J. Bacteriol. 154, 200, 1983), and at least four genes the hly A, hly B, hly C and hly D genes, are required to elicit a cell-free haemolytic phenotype. The hly C gene product appears to be required for activation of the hly A gene product which provides the haemolytically active species; whereas the hly B and hly D gene products (previously referred to as hly Ba and hly Bb) are essential for transport of haemolytic activity to the extracellular medium.
The primary product of the hly A gene does not contain an N-terminal signal signal sequence (Felmlee et al (1985) J. Bacteriol 163 p88-93). Thus, in an attempt to explain how secretion of the hly A gene product is achieved, Goebel and co-workers (Hartlein, M. et al, J. Cell Biochem. 22, 87-97, (1983)) have produced a model, in which the product of the hly A gene (107 Kd) is activated by the hly C gene product itself and is processed, possibly by an autoproteolytic activity located near the C-terminal end of the Hly A protein, to yield a smaller (58 Kd) haemolytically active peptide which is transported through the cytoplasmic membrane to the periplasm. They further propose that transfer of the 58 Kd haemolytically active peptide to the outer membrane and its release therefrom to the extracellular medium is promoted by the products of the hly B gene (46 Kd) and the hly D gene (62 Kd) respectively. Hartlein et al have also proposed the generation (by a proteolytic cut) of an N-terminal end of the 58 Kd fragment which can act as a signal peptide permitting transport of the fragment across the cytoplasmic membrane in the usual manner.
However, this explanation of the mechanism of secretion of the hly A gene product is controversial. Recently work in our laboratory and elsewhere has demonstrated the presence of large haemolytically active peptides, corresponding to the size of the initial hly A gene product, (107 Kd) in supernatants from cultures of haemolytic E. coli. This work suggests that cleavage of the Hly A protein is not required for production of the active extracellular haemolysin.
Hitherto, however, a satisfactory explanation of the mechanism by which haemolysin is secreted into the extracellular medium has not been forthcoming.
We have further studied the haemolysin secretion system and have shown that secretion of haemolysin from E. coli can be blocked by deletion of 27 amino acids from the C-terminus of the Hly A protein although this truncated molecule may be haemolytically activated by the hly C gene product. Further we have shown that a 23 Kd peptide from the C-terminus of Hly A contains all the information necessary for its own secretion. We have concluded, therefore, that all the information necessary for recognition and export of haemolysin in the presence of the hly B and hly D gene products, which appear to locate in the cell envelope, is contained within the 23 kilodalton C-terminal fragment of the hly A gene product. Our work constitutes the first finding of a C-terminal secretion sequence. Such C-terminal secretion sequences may be used in the preparation of recombinant fusion proteins which may be expressed and secreted from host cells.
The Hly B protein shows extensive homology (170 identical or conserved amino acid substitutions out of a stretch of 228 amino acids in the C-terminal region (Gerlach et al Nature 324 485-489 (1986)) with the p-glycoprotein Mdr (Multi-drug resistant protein) found in the surface of many drug resistant mammalian tumour cells. The Mdr protein is directly responsible for drug resistance and appears to function as an export pump (Ames, Cell 47 323-324 (1986)). The p-glycoprotein and haemolysin systems may therefore belong to a family of novel surface export mechanisms distinct from other export processes.